CDA/Cytidine deaminase

当吉西他滨和 ara-C 等细胞毒性脱氧胞苷类似物被分解代谢时，四氢尿苷 (THU)（胞苷脱氨酶 (CDA) 的一种特殊抑制剂）可防止脱氨。进行联合治疗以检查四氢尿苷对吉西他滨介导的肺癌细胞和胰腺癌细胞抗癌作用的影响。正如预期的那样，100 µM 四氢尿苷治疗后，BxPC-3 和 H441 中 CDA 表达升高，导致吉西他滨敏感性更高。 BxPC-3 和 H441 细胞系的敏感性分别提高了 2.1 倍和 4.4 倍。然而，尽管 MIAPaCa-2 和 H1299 细胞具有最低限度的 CDA 表达，但对吉西他滨表现出惊人的高敏感性。 MIAPaCa-2 和 H1299 细胞的 IC50 分别表现出 2.2 倍和 2.3 倍的变化。然而，Panc-1 和 H322 细胞的药物敏感性没有表现出任何明显的改变。这些发现表明，与 CDA 表达水平无关，四氢尿苷使一些肺癌和胰腺癌细胞对吉西他滨诱导的细胞死亡敏感。在不引发细胞凋亡的情况下，四氢尿苷抑制 S 期 [1]。

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四氢尿苷（THU）是一种有效的胞苷脱氨酶（CDA）抑制剂。高表达的CDA催化胞苷类似物并使其失活，最终导致吉西他滨耐药性增加。因此，四氢大麻素和吉西他滨的联合治疗被认为是治疗高表达CDA肿瘤的一种有潜力和前景的治疗方法。在本研究中，我们发现THU具有一种不依赖于CDA表达的抑制细胞生长的机制。三种不同的癌细胞系（MIAPaCa-2， H441和H1299）在单独给药THU后表现出细胞增殖下降，而敲除CDA则不受影响。为探讨thu诱导的细胞生长抑制机制，采用流式细胞术进行细胞周期分析。分析表明，THU使g1期发生率升高，而s期发生率降低。同样，Ki-67染色进一步支持THU降低细胞增殖。我们还发现THU通过抑制E2F1在G1/S检查点调节细胞周期进程。因此，THU和吉西他滨联合治疗胰腺癌可能比以前认为的更有效，因为THU作为CDA抑制剂，以及某些类型胰腺癌细胞的细胞生长抑制剂。［１］

一只雄性和八只雌性动物在接受 167 mg/kg 四氢尿苷 (THU) 和 1.0 mg/kg DAC 后死亡。在计划终止后幸存的动物大多没有症状，并且在接受高达 1.0 mg/kg DAC 和 167 mg/kg 四氢尿苷治疗的动物中，体重、食物消耗、临床化学或尿液分析没有变化[2]。

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Decitabine (5-aza-2脱氧胞苷;DAC)联合四氢尿苷（THU）是一种治疗镰状细胞病和β-地中海贫血的潜在口服疗法。在小鼠中进行了一项研究，以评估这种联合治疗的安全性，方法是在DAC前1小时口服DAC和THU，连续2天/周，持续9周，然后恢复28天，以支持其临床试验长达9周的持续时间。四氢尿苷，胞苷脱氨酶的竞争性抑制剂，被用于联合提高DAC的口服生物利用度。剂量为167 mg/kg THU，随后是0,0.2,0.4或1.0 mg/kg DAC；THU载药后加入1.0 mg/kg DAC；或者车辆本身。评估的终点是临床观察、体重、食物消耗、临床病理、大体/组织病理、骨髓微核和毒性动力学。在体重、食物消耗、血清化学或尿液分析参数方面没有发现与治疗相关的影响。血浆DAC水平的剂量和性别依赖性变化在1小时内达到Cmax。在测试的1mg /kg剂量下，与单独使用DAC相比，THU增加了DAC的血浆浓度（~ 10倍）。接受高剂量1 mg/kg DAC + THU的女性出现严重毒性，需要在第5周停止治疗。显微镜检查结果的严重程度和发生率呈剂量依赖性增加；结果包括骨髓细胞减少（伴随相应的血液学改变，白细胞、红细胞、血红蛋白、红细胞压积、网状细胞、中性粒细胞和淋巴细胞减少）、胸腺/淋巴细胞减少、肠上皮细胞凋亡和睾丸变性。骨髓微核分析证实骨髓细胞毒性，抑制红细胞生成和遗传毒性。在恢复期之后，观察到这些影响完全或有缓解的趋势。总之，联合治疗导致DAC毒性敏感性增加，与DAC血浆水平相关，女性比男性更敏感。[2]

Ki-67染色[1]
为了进行免疫细胞化学（ICC），将细胞以20-30%的合流度接种在室内载玻片中。孵育12 h后，加入100µM tetrahydrouridine (THU)孵育4 d。用4%多聚甲醛将细胞样品固定在PBS中15分钟后，进行ICC程序。为了使样品具有渗透性，将样品在含有0.3% Triton X-100的PBS中孵育10分钟。细胞用含有1% BSA的PBS阻断液处理60分钟。样品与Ki-67抗体在阻断缓冲液中孵育90分钟，然后用山羊抗兔二级抗体孵育60分钟。载玻片用链亲和素-生物素复合物试剂处理30分钟，用0.4%二氨基联苯胺(DAB)/H2O2孵育。染色过程的所有阶段都在室温下进行，载玻片在PBS中洗涤。停止DAB反应后，将载玻片安装在光镜下分析。在全细胞面积400倍放大下，计数1000个细胞中所有阳性反应，评价ICC染色效果。

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胞苷脱氨酶-siRNA [1]
瞬时转染 为了降低CDA在MIAPaCa-2、H441和H1299细胞中的表达，将所有细胞以70-80%的合流度接种于6孔板中。两个胞苷脱氨酶和一个阴性对照sirna从商业上购买。用Lipofectamine 2000在每个细胞系中分别转染所有sirna，最终浓度为每孔60 nM。为了测量mRNA水平，将细胞孵育48小时，然后收获用于实时PCR分析。转染12小时后，按照上述程序大纲测定细胞生长情况。我们还使用增殖试验测试了CDA抑制加tetrahydrouridine (THU)处理是否超过THU单独处理的效率。转染后的细胞在96孔板中接种过夜，加入100µM的THU。如上所述，在第0、2、4天测量OD。

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免疫印迹分析[1]
为了评估tetrahydrouridine (THU)是否在蛋白水平上影响细胞周期相关因子，我们进行了western blotting。加入100µM THU或PBS作为对照4天后，用含有蛋白酶抑制剂的RIPA缓冲液从细胞颗粒中提取蛋白质。采用Bio-Rad蛋白测定液测定蛋白浓度。在还原条件下，用SDS-PAGE凝胶分离等量的总蛋白。然后将蛋白质转移到聚偏二氟乙烯（PVDF）膜上，然后在TBS-Tween中用5%脱脂牛奶阻断。然后用抗E2F1和β-肌动蛋白的一抗按1∶1000稀释孵育膜。采用辣根过氧化物酶（HRP）偶联二抗，稀释倍数为1∶5000。使用SuperSignal West化学发光溶液实现可视化。

吉西他滨化学敏感性tetrahydrouridine (THU) [1]
药物敏感性试验基本上按照以前的报告所述进行。细胞接种于96孔板中，2500 ~ 5000个细胞/孔，一式三次。孵育12小时后，用4倍连续稀释的吉西他滨（从100µM开始）处理细胞，在37℃加或不加THU孵育96小时，以25%戊二醛在室温下固定30分钟，然后用200µl 0.05%亚甲基蓝染色20分钟，以0.33 M HCl搅拌洗脱染料20分钟，测定细胞活力。在598 nm处用酶标仪测定吸光度。计算50%细胞生长抑制浓度（IC50）。

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细胞生长试验[1]
采用亚甲基蓝比色法在96孔板中以5000个细胞/孔的密度进行胰腺癌和肺癌细胞系的细胞生长。细胞分别暴露于或未暴露于tetrahydrouridine (THU)，前12小时为第0天。从三个不同的井中分三次计算平均值，持续四天。

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流式细胞术分析细胞周期[1]
为了用流式细胞术分析细胞周期，将细胞以60%的合流度接种在10厘米的培养皿中。12 h后，每个培养皿中加入100µMtetrahydrouridine (THU)，连续3天。然后用胰蛋白酶- edta(胰蛋白酶0.25%；EDTA 0.02%)在37℃下静置2 ~ 5min，转移至15ml管中，PBS洗涤三次。然后将细胞在4°C 70%乙醇中固定过夜，然后用PBS再次洗涤，并在PBS-triton- x100（0.1%）中重悬。同时用RNaseA处理细胞，并用碘化丙啶染色。使用FACS口径流式细胞仪测定细胞周期状态，并使用FlowJo-887软件进行分析。

Experimental Design [2] Mice were assigned to four dose groups and a vehicle control group as shown in Table 1. Animals were gavaged with Decitabine (5-aza-2'-deoxycytidine; DAC) or its vehicle 1 hour ± 5 minutes after administration of tetrahydrouridine (THU) or its vehicle at a dose volume of 10 mL/kg. The DAC doses were selected based on the range finding study in which the mice tolerated six oral doses (2x/week) of 0.1, 0.2 and 0.4 mg/kg DAC in combination with a fixed dose of 167 mg/kg tetrahydrouridine (THU). A fixed THU dose (500 mg/m2) and the optimal timing between THU and DAC administration (60 min) were selected based on previous studies11. Conversion of milligrams per body surface area dose in mice into milligrams per kilogram body weight dose estimation was based on Michaelis constant (km) values for mice obtained from US Food and Drug Administration published guidelines. In brief, the mouse dose in milligrams per body surface area (500 mg/m2) was divided by the km of 3 to convert the dose to milligrams per kilogram body weight (167 mg/kg). The working body weight range of mice in the guideline is 11-34 gram; the body weight range of mice used in this study was 24-38 gram.

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Toxicokinetics [2] Sample collection tubes were prepared prior to each collection day by adding 10 μL/tube of a 10 mg/mL tetrahydrouridine (THU) solution. Blood samples (~0.5 mL) were collected via intra-cardiac puncture from non-fasted, anesthetized toxicokinetic animals on study day 1 (Groups 2 to 5) and 58 (Groups 2 to 5 with the exception of Group 4 females) at 15, 30, 60, 90, 120 and 180 minutes after administration of Decitabine (5-aza-2'-deoxycytidine; DAC) from 3 animals/sex/group at each time point. Due to mortality in the Group 4 females, the first 5 surviving animals were necropsied on study day 38 and blood samples were collected from three females per time point at 15, 30, and 120 minutes following administration of DAC. All samples were collected within 5 minutes of the target time.

dog LD unreported >1 gm/kg Pharmaceutical Chemistry Journal, 10(423), 1976
monkey LD unreported >1 gm/kg Pharmaceutical Chemistry Journal, 10(423), 1976
dog LD intravenous >1 gm/kg National Technical Information Service., PB211-590

[1]. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels. PLoS One. 2012;7(5):e37424.

[2]. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85.

Tetrahydrouridine has been used in trials studying the treatment of Neoplasms, Lung Neoplasms, Breast Neoplasms, Sickle Cell Disease, and Head and Neck Cancer, among others.
Tetrahydrouridine is a synthetic pyrimidine nucleoside analogue with biomodulating activity. Tetrahydrouridine increases the efficacy of the radiosensitizer cytochlor (5-chloro-2'-deoxycytidine) by inhibiting the enzyme deoxycytidine monophosphate (dCMP) deaminase and preventing the premature deamination of the cytochlor metabolite 5-chloro-2'-deoxycytidine monophosphate (CldCMP) to 5-chloro-2'-deoxyuridine monophosphate (CldUMP); in turn, this increases tumor concentrations of CldUMP which is then further anabolized and incorporated selectively into tumor DNA as CldU (5-chloro-2'-deoxyuridine). (NCI04)
An inhibitor of nucleotide metabolism.

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Finally, a second chemotherapy regimen for advanced pancreatic carcinoma is a clinically relevant issue to be resolved urgently. In this study, we demonstrated that tetrahydrouridine (THU) acts on some tumor cell lines to be both A) independently cytotoxic and B) to sensitize gemcitabine cytotoxicity through E2F1 in both CDA-high and CDA-low expressing cells. Consequently, tetrahydrouridine (THU) could improve gemcitabine sensitivity for at least some gemcitabine resistant cells regardless of CDA expression. Importantly, tetrahydrouridine (THU) has been clinically available, has been established as safe, and is more reasonably priced than newly developed molecular targeted drugs. Further research is necessary to discover which cell types are appropriate for THU treatment to control proliferation. With improved THU stability in vivo, a combination treatment could be considered a more feasible and effective therapeutic approach than existing ones for advanced pancreatic carcinoma. [1]

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In summary, the combination therapy resulted in increased sensitivity to DAC-induced toxicity as compared to the DAC treatment alone, correlating with DAC plasma levels. DAC Cmax was in a range expected to cause cytotoxicity (> 0.5 μM) in all DAC treatment groups. Female mice had higher Cmax and are more sensitive compared to males. Furthermore, hematologic effects appeared to be the most sensitive safety biomarkers, suitable for monitoring in clinical trials. treatment-related effects resolved fully or showed a trend towards resolution by the end of the recovery period. An oral route of administration for this combination therapy appears to be promising for clinical evaluation in sickle cell disease and β-thalassemia. Thus, the current study in CD-1 mice yielded important toxicity parameters pertinent to potential therapeutic application of tetrahydrouridine (THU) and DAC combination oral therapy in human settings.[2]

C9H16N2O6

248.23314

248.1

C, 43.55; H, 6.50; N, 11.29; O, 38.67

18771-50-1

Tetrahydrouridine dihydrate;Tetrahydrouridine-d3

29243

White to off-white solid powder

1.6±0.1 g/cm3

668.6±55.0 °C at 760 mmHg

358.2±31.5 °C

0.0±4.6 mmHg at 25°C

1.632

-1.85

130.33

5

6

2

17

301

4

C1CN(C(=O)NC1O)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O

UCKYOOZPSJFJIZ-XVKVHKPRSA-N

InChI=1S/C9H16N2O6/c12-3-4-6(14)7(15)8(17-4)11-2-1-5(13)10-9(11)16/h4-8,12-15H,1-3H2,(H,10,16)/t4-,5?,6-,7-,8-/m1/s1

1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-hydroxytetrahydropyrimidin-2(1H)-one

THU, NSC 112907; NSC-112907; TETRAHYDROURIDINE; 18771-50-1; 3,4,5,6-Tetrahydrouridine; NSC-112907; 0NIZ8H6OL8; Uridine, 3,4,5,6-tetrahydro-; NSC-112907-D; U 23284; NSC112907.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。